Pre-mRNA splicing is an essential step in eukaryotic gene expression and serves as a key point of regulation. Splicing removes intervening intron sequences from precursors of messenger RNAs (pre- mRNA) to establish the correct reading frame for mRNA translation. Additionally, differential inclusion of exon coding sequences by alternative splicing vastly expands information potential of genes. Mutations that affect splicing are associated with a number of human diseases, including cancers. The goal of this proposal is to obtain and interpret structural information for the spliceosome, the very large macromolecular machine responsible for splicing catalysis. This information is necessary to understand how the spliceosome is able to precisely recognize very distant splice sites along a pre- mRNA and coordinate intron excision and exon ligation. Because the spliceosome is a dynamic complex composed of five structural RNAs (the U-rich small nuclear U1, U2, U4, U5 and U6 snRNAs) and over of 100 proteins, it presents special challenges to structural studies. We will combine sophisticated mass spectrometry approaches and electron microscopy studies to examine spliceosomes arrested at different stages relative to splicing chemistry. By comparing the different conformations, we will determine how the many spliceosome components are arranged and create models of spliceosome structure. These studies are critical to understanding mechanisms of splice site identification, spliceosome assembly, and splicing catalysis.